Biochemical characterization of cardiac α‐actin mutations A21V and D26N implicated in hypertrophic cardiomyopathy

Abstract

Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α‐actin are the cause in 1%–5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM‐linked cardiac α‐actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α‐actin variants and wild‐type protein revealed improper N‐terminal processing in the Spodoptera frugiperda (Sf‐9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N‐terminal deletion mutants lacking the N‐terminal cysteine (ΔC2). The ΔC2 wild‐type construct behaved similar to porcine cardiac α‐actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α‐actin. In vitro motility assays and transient‐kinetic studies probing the interaction of the actin variants with cardiac β‐myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac β‐myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild‐type and p.D26N to the same level of 1100 nm s−1.